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52 Cards in this Set
- Front
- Back
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What do small organic compounds (cofactors) do Example |
Increase the repertoire of reactions a protein can perform Catalyse reactions that none of the other 20amjnk acids can perform E.g. nad+ Nicotinamide adenine dinucleotide |
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What is the cofactor in haemoglobin and myoglobin |
Prosthetic heme group with an organic molecules and an iron ion |
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Define a prosthetic group |
Tightly bound metal/coenzyme possibly by a covalent bond to an enzyme |
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Haloenxyme equation |
Apoenzyme + cofactor = haloenzyme |
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Coenzymes can act as transient carriers of what |
Specific functional groups and are derived from vitamins |
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Define a vitamin |
Organic molecules required in small amounts in the diet as organism cannot synthesise them |
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What is nad+ derived from |
Vitamin niacin Humans can make niacin from trp but we don't have enough usually |
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Is the same coenzyme is used by two different enzymes what is this indicative of |
Similar catalytic mechanisms |
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What does nad+ do |
Electron carrier important in many enzymes accepts a hydride ion in the reactive nicotinamide ring R group is h in nad R group is p in nadp |
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What are metal ions main two functions |
Carriers of functional groups Form structural features |
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What do zinc fingers do |
Insert themselves into the major groove of DNA and regulate transcription 2beta, one alpha helix by coordination of a zinc to two his or two characters residues |
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How can zinc fingers be predicted |
4his/cys spaced appropriately in the primary structure |
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Do zinc fingers actually bind to dna |
Zinc fingers stabilise an elongated loop of approx 30 residues, these amino acids specify dna interactions and often bind to the major groove of dna |
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How else can the function be increase past the 20 amino acids |
Covalent modification Glycosylation Hydroxylation Phosphorylation Acetylation |
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What is glycosylation |
Adding sugars(carbohydrates) to make the protein more hydrophilic, more soluble and stable in aqueous solution |
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What three ways is glycosylation used in the proteins |
By cell surface proteins on bacteria to evade antibodies produced by immune system by changing rapidly between generations Bacterial pathogenesis Labels targeting proteins in specific locations e.g. organelles for destruction Provide information of cell surfaces Cell cell recognition |
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What is o linked glycosylation |
Glycosidic bond to hydroxyl if set/thr Galnac(n-acetylgalactosamine) |
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What is n linked glycosylation |
N glycosyl bind to n of asn glcnac(n-acetylglucosamine) |
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How can n linked glycosylation be predicted |
Asn-X-SER/THR when x is any amino acid but not proline |
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Are all n linked sites exploited |
Depends on factors such as accessibility and cell type |
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Is there a consensus for o linked glycosylation |
No |
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Why is hydroxylation and what amino acids can undergo this |
Pro/lys can have a hydroxyl group added Less common than glycosylation |
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What does hydroxylation do |
Increases h bonding potential and the range of side chains available in proteins |
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What does hydroxylation of outlines in collagen cause What is the cofactor needed |
Stability of the fibrillar structures as the oh locks the hydroxyproline into a specific conformation Prolyl hydroxylase is needed that requires vitamin c cofactor |
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What does a lack of vitamin c mean for collagen |
Insufficient proline hydroxylase working collage so reduces tissue strength and causes scurvy |
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What are the targets for phosphorylation and how are they phosphorylated |
Ser/thr/tyr/his targets P from ATP is added to hydroxyl of ser/the Added to n-h of his to add a negative charge (significant locally) to a moderately polar parts which can alter the conformation |
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What reverses phosphorylation and what enzyme causes it |
Reverse is protein phosphatases Cause is protein kinases Both needed to switch on and off |
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What happens when protein kinases mutate |
In many cancers this occurs, causes misregulation of the cell cycle so unrestricted cell division |
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How are protein kinases important in bacteria |
For cell signalling Sensor protein kinase is activated via a specific signal such as nutrient in an environment Kinase phosphorylates a second response regulator, often a tf that controls transcrip of specific set of genes needed to respond to the environmental signal |
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What is acetylation and what can be acetylated |
Lys via acetyl COA Adding a c2h6 group |
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What does acetylating lys cause |
Lys has a positive charge so there is an electrostatic attraction between it and dna, high affinity so sense packaging in nucleosome Acetylation opens chromatin so is accessible for transcription and tfs so controls gene transcription in eukaryotes |
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What enzymes are involved in acetylation |
transferanse Histone acetyltransferansehistone deacetylase histone deacetylase |
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What did properties are proteins purified based |
Solubility, size, charge, surface e hydrophobicity, binding affinity for a certain ligand |
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How does protein purification work if there are many with the same size |
Many have same size but few of these have the same charge |
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Proteins can be engineered with an affinity tag at the n or c terminus What is an affinity tag |
A peptide or polypeptide with a high binding affinity for a ligand |
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What does the histadine tag do and the nickel bead process of purification |
Multiples his residues that together can bind to ni2+ ions immobilised on a column matrix Fusing gene that codes for target protein to affinity tag When passed through beads, his tag binds so target protein has also bound specifically Very few proteins bind naturally so allows for a one stop process |
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If 99% purity is required how much does a 9% and a 0.09% total cell starting protein need to be scaled up |
11 and 1100 fold respectively |
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What are rich samples of protein that could be chose |
Animal, plant or environmental |
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What is a bacterial recombinant expression system |
Usually bacterial cells have been grown/ engineered to express a protein of interest Very rapid growth, large volumes and high cell density |
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What is the overall aim of protein purification |
Increase the specific activity of the target |
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Define activity |
Total units of enzyme in solution |
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Define specific activity |
Number of enzyme units per mg of total protein |
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How is an acceptable level of specific activity achieved |
Multiple purification steps |
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Why must yield and purity be balanced |
Each step loses some target protein Stop when specific activity is sufficient for application |
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Define yield |
Total activity of the targer |
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Define purity |
Specific activity of the target |
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Why is 100% purity impossible |
Contamination |
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What does an assay monitor |
Protein presence For an enzyme The accumulation of product or product depletion |
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Assay advantages |
Simple, quick and cheap |
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What is liquid chromatography |
Purification by passing a protein mix through a solid matrix usually of beads with an external aqueous environment Proteins washed off Proteins with higher affinity elite late and are detected by UV light |
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What is ion exchange chromatography |
Solid matrix that is neagtive so purifies positive proteins Elites by nacl/kcl in buffer Nacl at higher concentrations outcompete as the positive proteins in the matrix |
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What is size exclusion chromatography |
Pores are a specific size, larger proteins can't enter so elute faster Rods elute slower than globular of the same size Inert gel matrices so they don't separate base on charge Can estimate protein size by using proteins of a known molecular weight |
